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(Journal of Leukocyte Biology. 2003;73:344-355.)
© 2003 by Society for Leukocyte Biology

Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses

Mark K. Clements*, Daniel W. Siemsen*, Steve D. Swain*, Angela J. Hanson*, Laura K. Nelson-Overton*, Troy T. Rohn{dagger} and Mark T. Quinn*

* Department of Veterinary Molecular Biology, Montana State University, Bozeman; and
{dagger} Department of Biology, Boise State University, Idaho

Correspondence: Mark T. Quinn, Ph.D., Department of Veterinary Molecular Biology, Montana State University, P.O. Box 173610, Bozeman, MT 59717. E-mail: mquinn{at}montana.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite-mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN-1) modified actin in a concentration-dependent manner, resulting in an inhibition of globular-actin polymerization and filamentous-actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU-101033E, which has been shown previously to specifically block peroxynitrite-mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite-treated actin demonstrated a concentration-dependent increase in nitrotyrosine, which was also blocked by PNU-101033E. Activation of neutrophils in the presence of a nitric oxide donor (S-nitroso-N-acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite-treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin-dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN-1 treatment in a concentration-dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin-dependent, cellular processes in phagocytic cells and may modulate their host defense function.

Key Words: polymorphonuclear leukocytes • NADPH oxidase • nitrotyrosine • cytoskeleton


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear leukocytes or neutrophils play an essential role in the body’s defense against pathogenic microorganisms [1 ]. Upon exposure to a pathogen, these cells respond by producing a variety of reactive oxygen species (ROS), including superoxide anion (O2–), hydrogen peroxide, hypochlorous acid, and nitric oxide (NO) [2 , 3 ]. The concurrent production of NO and O2– at sites of inflammation can also result in the formation of secondary ROS such as peroxynitrite (rate constant 7x109 M-1s-1) [4 ]. Recent evidence has demonstrated that this reactive oxidant plays a key role in host defense [5 ] as well as in the pathogenesis of inflammatory disease [4 ].

The actual production of peroxynitrite by inflammatory cells in vivo has been demonstrated by activated rat alveolar macrophages [6 ], L-arginine-depleted mouse macrophage RAW 264.7 cells [7 ], and activated human neutrophils [8 , 9 ]. It is also likely that NO derived from endothelial cells reacts with neutrophil-generated O2– at sites of inflammation, resulting in peroxynitrite formation [10 ]. Irrespective of the source of NO, it is clear that significant concentrations of peroxynitrite can be generated in inflammatory tissues [4 ].

Peroxynitrite is a potent oxidant that can attack a wide variety of biological tissues, and recent research has implicated peroxynitrite as one of the damaging agents in a number of pathological, inflammatory conditions [4 ]. The cytotoxic action of peroxynitrite may be related to its ability to initiate lipid peroxidation [11 ], oxidize protein sulfhydryls [12 ], and nitrate tyrosine residues on a variety of proteins [13 ]. In relation to the latter action of peroxynitrite, it has been demonstrated that tyrosine nitration can lead to the inactivation of enzymes and/or receptors (reviewed in ref. [4 ]). For example, peroxynitrite has been shown to nitrate tyrosine residues in human manganese superoxide dismutase (SOD), resulting in inactivation of this enzyme [14 ]. Peroxynitrite can also modulate tyrosine-dependent signaling pathways in cells by blocking tyrosine kinase targets [15 ].

As a result of the highly reactive nature of peroxynitrite and its generation in close proximity to the neutrophil membrane, it seems likely that this ROS could have potential effects, cytotoxic or otherwise, on neutrophil function. In addition, as peroxynitrite has a relatively long half-life compared with other free radical species and is able to rapidly diffuse across biological membranes [16 ], membrane and cytosolic proteins would be potential targets for peroxynitrite attack. Recently, we found that peroxynitrite can modulate human neutrophil host defense responses [17 ], and that these effects were mediated primarily by nitration of tyrosine residues on neutrophil proteins. It is interesting that one of the most heavily nitrated proteins found in peroxynitrite-treated neutrophils had a molecular mass of ~45 kDa, the molecular weight range of actin [17 ]. As actin remodeling plays a key role in a number of neutrophil responses [18 , 19 ], we hypothesized that actin might be an important, functional target for peroxynitrite nitration.

In the present studies, we evaluated this hypothesis by investigating the effect of peroxynitrite on actin polymerization in vitro and in intact cells. We also analyzed the effects of peroxynitrite on neutrophil functions where actin polymerization has been implicated, including respiratory burst, chemotaxis, and phagocytosis. In in vitro and intact cell systems, peroxynitrite-mediated tyrosine nitration resulted in inhibition of actin polymerization. Analysis of peroxynitrite-treated neutrophils demonstrated that actin was indeed a major target for nitration in intact cells. Furthermore, a range of actin-dependent neutrophil functional responses was inhibited by peroxynitrite. Thus, the ability of peroxynitrite to modulate neutrophil actin polymerization through actin nitration further confirms that this ROS plays an important role in modulating neutrophil function during inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Sodium peroxynitrite (>90% pure with the balance being nitrate/nitrite) was purchased from Cayman Chemical (Ann Arbor, MI). 3-Morpholinosyndonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), xanthine (X), xanthine oxidase (XO), and SOD were purchased from Sigma Chemical Co. (St. Louis, MO). Leukotriene B4 (LTB4), recombinant human interleukin-8 (IL-8), and Pansorbin cells were purchased from Calbiochem (La Jolla, CA). Rabbit anti-nitrotyrosine polyclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-actin antibody was obtained from Amersham Biosciences (Piscataway, NJ). Alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG) and an alkaline phosphate substrate kit were purchased from BioRad (Hercules, CA). Antioxidant PNU-101033E, obtained from Pharmacia & Upjohn Co. (Kalamazoo, MI), was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at -20°C. On the day of the experiment, the stock solution was serially diluted in DMSO to achieve the desired drug concentration. For all samples where PNU-101033E was tested, an equal volume of DMSO was tested as a vehicle control.

Actin preparation
Actin acetone powder was prepared from rabbit skeletal muscle and extracted twice with buffer A [2 mM Tris, pH 8.0, 0.2 mM adenosine 5'-triphosphate (ATP), 0.2 mM CaCl2, 0.5 mM ß-mercaptoethanol, 0.005% NaN3] as described by Pardee and Spudich [20 ]. Preparations of actin were depolymerized by dialysis against buffer A for the first 24 h followed by dialysis in buffer A without ß-mercaptoethanol. Monomeric globular actin (G-actin) was then centrifuged for 1 h at 125,000 g at 4°C, and the protein concentration was determined using the BCA protein assay (Pierce Chemical Co., Rockford, IL).

Pyrene actin was prepared by labeling actin with N-(pyrenyl)iodoacetamide (Molecular Probes, Eugene, OR) as described by Cooper et al. [21 ]. Pyrene-labeled G-actin was prepared by dialysis in buffer A as described above.

Actin polymerization assay
Pyrene actin and unlabeled actin were mixed 1:1 and diluted to a final concentration of 80 µg/ml in buffer A ± 25 mM NaHCO3, and 200 µl of the mixture was aliquotted into 96-well FluoroNunc microtiter plates (Nalge Nunc International, Rochester, NY). Baseline fluorescence was monitored for 5 min at 37°C using a Fluoroscan fluorescence microtiter plate reader (Labsystems, Helsinki, Finland) with {lambda}excitation at 355 nm and {lambda}emission at 405 nm. Polymerization was initiated by the addition of 2 mM MgCl2 (2µl of a 0.2 M stock), followed by mixing for 10 s. Actin polymerization, as determined by an increase in pyrene fluorescence, was monitored for 20 min.

In peroxynitrite-treated samples, actin was treated before aliquotting into microtiter plates. For treatment with authentic peroxynitrite, serial dilutions of peroxynitrite were prepared in 0.3 M NaOH from 175 mM stock peroxynitrite, and 2 µl was added per ml pyrene actin mixture, followed by the addition of 2 µl 0.3 M HCl to neutralize the pH. The samples were analyzed immediately after peroxynitrite treatment. For SIN-1 treatment, serial dilutions of SIN-1 were prepared in buffer A (starting stock was 50 mM), and 100 µl was added per ml pyrene actin mixture. The samples were incubated with SIN-1 for 1 h with gentle shaking and analyzed as above. For samples containing the antioxidant PNU-101033E (100 µM), controls included a similar volume of DMSO (vehicle control).

Actin depolymerization assay
Pyrene actin and unlabeled actin were mixed 1:1 and polymerized overnight at 4°C by adding 50 mM KCl and 2 mM MgCl2. Filamentous actin (F-actin) was then diluted to a final concentration of 80 µg/ml in buffer A or buffer A with 25 mM NaHCO3, as indicated. F-actin samples (200 µl/well) were aliquoted into microtiter plates, and pyrene fluorescence was monitored for 1 h, as described above. For SIN-1-treatment, 20 µl SIN-1 (0–5 mM final concentrations) was added to each well before analysis. Where indicated, 100 µM PNU-101033E was included as described above.

Spectrophotometric detection of nitrotyrosine
Actin was treated with the indicated concentrations of peroxynitrite or SIN-1, as described above, and analyzed for the presence of nitrotyrosine using a modification of the method described by Gow et al. [22 ]. After peroxynitrite or SIN-1 treatment, the pH was adjusted to 11.5, aliquots were placed in microtiter plates, and absorbance was measured at 430 nm (the absorbance maximum at this pH; ref. [22 ]) using a VERSAMax microtiter plate reader (Molecular Devices, Sunnyvale, CA). The amount of nitrotyrosine present in the protein was calculated using {varepsilon}430 nm= 4400 M-1cm-1 [23 ]. For experiments testing, the effect of bicarbonate, 25 mM NaHCO3, was added to the reaction mixture before peroxynitrite treatment, and the pH was readjusted to 7.4. Where indicated, 100 µM PNU-101033E was included as described above.

Measurement of dityrosine
Actin (0.5 mg/ml) was treated with control buffer or the indicated concentrations of SIN-1 in buffer A ± NaHCO3, and dityrosine formation was monitored by fluorescence measurements on a Perkin Elmer LS-50B scanning fluorimiter using {lambda}excitation at 325 nm and {lambda}emission at 415 nm, as described by Davies et al. [24 ].

Measurement of actin carbonylation
Actin (0.5 mg/ml) was treated with control buffer or the indicated concentrations of SIN-1 in buffer A ± NaHCO3, and carbonyl formation was determined by incubation with an equal volume of 10 mM 2,4-dinitrophenylhydrazine in 2 M HCl in the dark at room temperature for 1 h with vortexing, as described by Dalle-Donne et al. [25 ]. Samples were precipitated with 20% trichloroacetic acid, washed with ethanol:ethylacetate (1:1), resuspended in 1 ml 6 M guanidine hydrochloride dissolved in 20 mM sodium phosphate, pH 2.3, and warmed to 37°C for 15 min. The absorbance was monitored at 366 nm.

Neutrophil isolation and peroxynitrite treatment
Neutrophils were purified from human blood using dextran sedimentation followed by hypotonic lysis of red blood cells and Histopaque 1077 gradient separation, as described previously [26 ]. Cell preparations were routinely >95% pure, as determined by light microscopy, and >98% viable, as determined by trypan blue exclusion.

Purified neutrophils were resuspended (107/ml) in Hanks’ balanced salt solution (HBSS) and treated for 1–2 h at room temperature with the indicated concentrations of SIN-1. Control cells were incubated with a similar volume of buffer instead of SIN-1.

Immunoprecipitation
Immunoprecipitation was performed following methods of DeLeo et al. [27 ]. Briefly, neutrophils were treated for 1 h at room temperature with 5 mM SIN-1, placed on ice, and incubated for 15 min with 3 mM diisopropylfluorophosphate (DFP). The cells were washed, resuspended in HBSS containing 1% Triton X-100 and protease inhibitor cocktail (Sigma Chemical Co.), lysed for 1 h on ice, and centrifuged to remove insoluble material (15 s at 12,000 rpm). The supernatant was precleared with 50 µl/ml nonimmune rabbit serum (rotation at 4°C for 1 h), followed by 50 µl/ml washed Pansorbin cells (rotation at 4°C for an additional 1 h), followed by centrifugation to remove immune complexes. The precleared supernatant was immunoprecipitated with preimmune serum (control) or antinitrotyrosine antibody affinity-purified from antinitrotyrosine antiserum with nitrotyrosine-conjugated Sepharose [23 ]. Reactions were rotated overnight at 4°C, Protein A Sepharose (Amersham Biosciences) was added, and samples were rotated an additional 1 h at 4°C. The beads were washed 5x, resuspended in sample buffer, boiled, and analyzed by electrophoresis and Western blotting, as described below.

Nitration of exogenous actin by activated neutrophils
Purified neutrophils (108/ml) were resuspended in HBSS containing 100 µg/ml actin ± 5 mM SNAP and stimulated for 5 min at 37°C with 1 µg/ml phorbol myristate acetate (PMA). The samples were placed on ice, treated for 15 min with DFP to inactivate proteases, and then centrifuged to pellet the cells. The supernatant was collected, solubilized in sample buffer, and analyzed for nitrated actin by Western blotting, as described below.

Neutrophil actin polymerization
Changes in the content of neutrophil F-actin were determined by using BODIPY-phallacidin, a fluorescent F-actin-binding probe following the method of Mineshita et al. [28 ]. Briefly, control and SIN-1-treated cells (107/ml; prepared as described above) were warmed to 37°C in a water bath and treated with 10-8 M IL-8 at for 0, 30, or 60 s. After treatment, cells were fixed in Dulbecco-Vogt phosphate-buffered saline (DPBS) containing 3.7% formaldehyde for 15 min at room temperature. The cells were washed and stained at 37°C with 165 nM BODIPY-phallacidin (Molecular Probes) + 110 µg/ml lysophosphatidylcholine (Avanti Polar Lipids, Inc., Alabaster, AL) at 37°C for 15 min. Cells were washed and analyzed by flow cytometry on a FACSCalibur.

Neutrophil migration
Migration of control and SIN-1-treated cells (treated as described above) was measured in Falcon 24-well plates containing transwell inserts, as described previously [29 , 30 ]. Briefly, transwell inserts containing 2.5 x 105 neutrophils suspended in HBSS + 0.1% bovine serum albumin were placed in wells containing buffer (control) or chemoattractant (10-7 M LTB4). After incubation for 1 h at 37°C in a humidified 5% CO2 incubator, the transwell inserts were removed, and the number of cells migrated to the lower wells was determined using a lactate dehydrogenase (LDH) assay (CytoTox 96 nonradioactive cytotoxicity assay, Promega, Madison, WI). LDH measurements were then converted to percent-cell migration by comparison of the values with standard curves of LDH measurements from known cell concentrations.

Phagocytosis and bacterial killing
A Vybrant phagocytosis assay kit (Molecular Probes) was used to measure neutrophil phagocytosis of control and SIN-1-treated cells. Briefly, 3 x 105 cells were incubated for 1 h at 37°C with fluorescein-labeled Escherichia coli (K-12 strain) or Staphylococcus aureus BioParticles. The cells were washed, resuspended in HBSS without Ca2+, and incubated an additional 5–10 min to allow internalization of bound bacteria. The cells were then washed, incubated for 5 min at room temperature in trypan blue solution, washed again, and analyzed by flow cytometry on a FACSCalibur using the FL1 channel. The level of phagocytosis is expressed as relative fluorescence units.

Analysis of bacterial killing was performed as described by Hampton et al. [31 ]. Briefly, S. aureus was opsonized for 20 min at 37°C DPBS containing 1 mg/ml glucose and 10% normal human serum (NHS). Neutrophils and opsonized S. aureus were mixed at a ratio of 1:1 (107 each) in DPBS/glucose/10% NHS and incubated at 37°C with rocking. At the indicated times, aliquots were removed, the total number of viable bacteria present outside and inside the neutrophils was measured, and the number of bacteria killed was determined by adjusting for bacterial growth measured in control samples containing bacteria alone and incubated for the same times [31 ].

Neutrophil superoxide generation
O2– production by control and SIN-1-treated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLF) was determined by measuring the rate of SOD-inhibitable ferricytochrome c reduction as described previously [26 ]. Briefly, the cells (2x105) were added to microtiter plate wells containing DPBS, 1 mM CaCl2, and 100 µM cytochrome c. After addition of 10-7 M fMLF, absorbance at 550 nm was monitored continuously for 15 min at 37°C with mixing between measurements in a THERMOmax microtiter plate reader (Molecular Devices). Duplicate wells containing 80 µg/ml SOD were included to determine nonspecific reduction of cytochrome c. Rates of SOD-inhibitable O2– production were calculated using {varepsilon} = 21 x 103 M-1cm-1 for cytochrome c.

Electrophoresis and Western blotting
Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7–18% polyacrylamide gradient gels and were transferred to nitrocellulose, as described previously [32 ]. Prestained molecular weight standards (BRL, Bethesda, MD) were included on all gels for reference. Actin blots were probed with monoclonal antiactin antibody followed by alkaline phosphatase-conjugated goat anti-mouse IgM secondary antibody (Jackson Immunoresearch, West Grove, PA) at a 1:1000 dilution and were developed using an AP conjugate substrate kit (BioRad, Richmond, CA). Nitrotyrosine blots were probed with monoclonal antinitrotyrosine antibody (Alexis Biochemicals, San Diego, CA), followed by horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (BioRad, Richmond, CA) at a 1:20,000 dilution and were developed using a SuperSignal West Pico chemiluminescent substrate kit (Pierce) and BioMax MR film (Eastman Kodak Co., Rochester, NY). Image analysis was performed with an IS-1000 Alpha Imager imaging system (Alpha Innotech, San Leandro, CA).

Statistical analysis
Data were analyzed with one-way ANOVA, and statistical differences between groups were determined by Newman-Keuls multiple comparison test (GraphPad Prism, San Diego, CA). As a minimum, P< 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of peroxynitrite on actin polymerization in vitro
Exposure of G-actin to increasing concentrations of peroxynitrite resulted in a concentration-dependent inhibition of the rate of actin polymerization, with an inhibitory concentration (IC50) of ~45 µM (Fig. 1 , upper panel). Decomposed peroxynitrite (10 min at room temperature and pH 7.4) had no effect (not shown). Consistent with the ability of peroxynitrite to nitrate protein tyrosine residues, exposure of G-actin to peroxynitrite resulted in a concentration-dependent increase in nitrotyrosine in the treated actin, as determined by monitoring absorbance at 430 nm (Fig. 1 , lower panel) and by Western blot analysis (Fig. 2 ). Note that increased nitrotyrosine staining on the blots was not a result of loading differing amounts of actin, as actin immunoblots showed equal loading of actin in all lanes (Fig. 2) . Furthermore, actin tyrosine nitration was increased by the addition of 25 mM NaHCO3, which has been shown to enhance peroxynitrite-mediated tyrosine nitration at physiological pH [22 ] and was completely blocked by the addition of a pyrrolopyrimidine antioxidant (100 µM PNU-101033E), which has been shown to prevent protein tyrosine nitration by peroxynitrite [33 ] (Fig. 1 , lower panel).



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  		Figure 1. Inhibition of actin polymerization by peroxynitrite. (Upper) G-actin was treated with the indicated concentrations of authentic peroxynitrite, the sample pH was neutralized, and the rate of actin polymerization was measured as described. The results are expressed as the % of control actin polymerization rate. (Lower) G-actin was treated with the indicated concentrations of authentic peroxynitrite in buffer A (open bars), buffer A + 25 mM NaHCO3 (hatched bars), or buffer A + 100 µM PNU-101033E (solid bars), and the amount of nitrotyrosine was determined spectrophotometrically as described. In both panels, the results are expressed as mean ± SEM and represent three independent experiments. Statistically significant differences (#, P<0.01; *, P<0.001) between control (0) and peroxynitrite-treated samples are indicated.

 


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  		Figure 2. Nitrotyrosine formation in peroxynitrite-treated actin. G-actin was treated with 0, 5, 10, 20, 40, 75, 150, and 300 µM authentic peroxynitrite, as described in Figure 1 (lanes 1–8, respectively). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-actin to show consistent loading of actin (upper) and anti-nitrotyrosine (lower) antibodies as indicated. Lanes containing prestained molecular weight markers are shown (STD). Note that the STD locations on nitrotyrosine blots were inscribed with a marker. The data are representative of three independent experiments.

 
Treatment with peroxynitrite requires the addition of a bolus of peroxynitrite, followed by neutralization of the sample, and this may not accurately reflect the physiological situation, where lower levels of peroxynitrite are generated over a longer period of time. Therefore, we performed similar actin polymerization studies in the presence of a peroxynitrite generator (SIN-1) [34 ]. Consistent with our results using bolus peroxynitrite, exposure of G-actin to SIN-1 also resulted in a concentration-dependent inhibition of actin polymerization (Fig. 3 , upper panel) with a concomitant concentration-dependent formation of nitrotyrosine (Figs. 3 , lower panel, and 4 ). Again, the presence of NaHCO3 substantially enhanced the inhibitory effect (Figs. 3 and 4) , supporting a role for tyrosine nitration rather than other types of protein modification [35 ]. Furthermore, the inhibition of actin polymerization and the nitration of actin tyrosine residues resulting from SIN-1 treatment were largely prevented by the addition of PNU-101033E (Figs. 3 and 4 ). By comparison, analysis of SIN-1-treated actin for the presence of dityrosine or carbonyl groups showed little or no dityrosine or carbonyl formation at any of the SIN-1 concentrations tested (0–5 mM; data not shown), indicating the effects on actin polymerization were primarily a result of tyrosine nitration and not the formation of dityrosine cross-links or modification by carbonyl groups.



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  		Figure 3. Inhibition of actin polymerization by SIN-1. (Upper) G-actin was treated with the indicated concentrations of SIN-1 in buffer A ({square}), buffer A + 25 mM NaHCO3 (•), or buffer A + 100 µM PNU-101033E ({blacktriangleup}), and the rate of actin polymerization was measured as described. The results are expressed as the % of control actin polymerization rate. (Lower) G-actin was treated with the indicated concentrations of SIN-1 in buffer A (open bars), buffer A + 25 mM NaHCO3 (hatched bars), or buffer A + 100 µM PNU-101033E NaHCO3 (solid bars), and the amount of nitrotyrosine was determined spectrophotometrically as described. In both panels, the results are expressed as mean ± SEM and represent five independent experiments. Statistically significant differences (#, P<0.01; *, P<0.001) between control (0) and SIN-1-treated samples are indicated.

 


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  		Figure 4. Nitrotyrosine formation in SIN-1-treated actin (polymerization assay). G-actin was treated with 0, 0.08, 0.16, 0.31, 0.62, 1.25, 2.5, and 5.0 mM SIN-1, as described in Figure 3 (lanes 1–8, respectively). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-actin to show consistent loading of actin (upper panels) and anti-nitrotyrosine (lower panels) antibodies as indicated. Lanes containing prestained molecular weight markers are shown (STD). Note that the STD locations on nitrotyrosine blots were inscribed with a marker. The data are representative of five independent experiments.

 
The effect of peroxynitrite treatment on F-actin filaments was also investigated. It is interesting that SIN-1 treatment induced a concentration-dependent depolymerization of actin filaments, which was enhanced by the presence of NaHCO3 and inhibited by PNU-101033E (Fig. 5 ). We also observed a concentration-dependent formation of nitrotyrosine, which was enhanced by the presence of NaHCO3 and inhibited by PNU-101033E (Fig. 6 ). Thus, nitration of actin tyrosine residues by peroxynitrite not only can inhibit G-actin polymerization but can also induce depolymerization of F-actin filaments.



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  		Figure 5. Inhibition of actin depolymerization by SIN-1. F-actin was treated with the indicated concentrations of SIN-1 in buffer A (open bars), buffer A + 25 mM NaHCO3 (hatched bars), or buffer A + 100 µM PNU-101033E (solid bars), and the amount of F-actin was measured as described. The results are expressed as the % of control F-actin remaining after treatment (mean±SEM) and represent three independent experiments. Statistically significant differences (#, P<0.01; *, P<0.001) between control (0) and SIN-1-treated samples are indicated.

 


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  		Figure 6. Nitrotyrosine formation in SIN-1-treated actin (depolymerization assay). F-actin was treated with 0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, and 5.0 mM SIN-1, as described in Figure 5 (lanes 1–8, respectively). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-actin to show consistent loading of actin (upper panels) and anti-nitrotyrosine (lower panels) antibodies as indicated. Lanes containing prestained molecular weight markers are shown (STD). Note that the STD locations on nitrotyrosine blots were inscribed with a marker. The data are representative of three independent experiments.

 
Actin nitration in SIN-1-treated neutrophils
Based on the data reported here and our previous studies showing that peroxynitrite treatment of neutrophils resulted in nitration of several proteins, including proteins at ~45 kDa [17 ], we hypothesized that actin might be a key target for peroxynitrite that would contribute to the effects on neutrophil function observed after peroxynitrite treatment [17 ]. To address this, neutrophils were treated with SIN-1, and nitrated proteins were analyzed by Western blotting. Although a number of proteins were nitrated in SIN-1-treated neutrophils, the most heavily nitrated protein was located at 42–43 kDa, which is identical to the molecular mass of actin (Fig. 7 ). Immunoblotting for actin demonstrated the presence of actin at the same location on the blots as the heavily nitrated protein, suggesting that actin was nitrated in SIN-1-treated cells (Fig. 7A) . Furthermore, we immunoprecipitated SIN-1-treated neutrophil lysates with affinity-purified antinitrotyrosine antiserum and found that actin was indeed immunoprecipitated (Fig. 7B) , thereby confirming that actin represents one of the major targets for nitration during exposure of neutrophils to peroxynitrite. Note that an abundant protein of 85–90 kDa was also present in the immunoprecipitates; however, comparison of nitration (Fig. 7A) suggests this protein was not nitrated nearly as much as actin. Currently, the identity of this protein is unknown.



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  		Figure 7. Nitration of actin in intact neutrophils. (A) Purified human neutrophils (106 cells/ml) were treated with buffer (lane 1) or 5 mM SIN-1 (lane 2) for 1 h at room temperature and were then solubilized in SDS-PAGE sample buffer. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with IgM anti-actin (Actin) and IgG anti-nitrotyrosine (NT) antibodies, as indicated. The locations of molecular weight markers are shown. (B) Neutrophils (107 cells/ml) were treated with 5 mM SIN-1 for 1 h at room temperature and solubilized in HBSS + 1% Triton X-100 and protease inhibitors, as described. Neutrophil lysates (starting material shown in lanes 1; lanes indicated for gel and blot) were immunoprecipitated with preimmune serum (lanes 2) or affinity-purified anti-nitrotyrosine antibody (lanes 3). The samples were separated by SDS-PAGE and analyzed by silver staining and immunoblotting with anti-actin antibodies, as indicated. Lanes containing prestained molecular weight markers are shown on the gel (STD). Locations of the IgG heavy (H) and light (L) chains are indicated; representative of three independent experiments.

 
Nitration of actin by activated neutrophils
To evaluate the potential of neutrophils to nitrate actin during the inflammatory response in vivo, we activated neutrophils in suspensions containing exogenous actin. In these experiments, we found that activation of human neutrophils in the presence of the NO generator SNAP resulted in the formation of sufficient peroxynitrite to nitrate exogenously added actin (Fig. 8 ). Actin was nitrated in samples containing PMA-activated neutrophils and SNAP (lanes 3) but not in samples containing activated neutrophils without SNAP (lanes 2). The requirement for SNAP in these studies is consistent with previous studies by Grisham and coworkers [36 ], who reported that fully activated neutrophils produce very little peroxynitrite in vitro. They concluded that the relative ratio of these two radicals played a key role in controlling the steady-state concentration of peroxynitrite [36 , 37 ]. Optimally, equimolar fluxes of NO and O2– react to yield peroxynitrite; however, excess production of either radical is inhibitory [37 ]. As activated neutrophils generate far more O2– than NO, the addition of SNAP serves to equalize the O2–-to-NO ratio, resulting in an enhanced generation of peroxynitrite, which would be more representative of the site-specific generation of these oxidants that could occur in inflamed tissues.



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  		Figure 8. Nitration of exogenous actin by activated neutrophils. Purified actin (100 µg/ml) was incubated for 5 min at 37°C with buffer alone (lanes 1), buffer containing activated (1 µg/ml PMA) human neutrophils (lanes 2), and buffer containing activated human neutrophils + 5 mM SNAP (lanes 3), as described. The samples were placed on ice, treated with DFP, and centrifuged to pellet the cells. The supernatants were collected, solubilized in sample buffer, and analyzed for nitrotyrosine and actin, as indicated, by Western blotting. Representative of three independent experiments.

 
Effects of SIN-1 on actin-dependent neutrophil functional responses
Previous studies have demonstrated that actin polymerization is an important component of neutrophil activation and priming responses [18 , 19 ] and that association with the actin cytoskeleton plays a role in regulation of chemoattractant receptor and adhesion protein function [38 , 39 ]. In addition, the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to be associated with the actin cytoskeleton [40 ]. Therefore, we analyzed the effects of SIN-1-generated peroxynitrite on several key actin-dependent, functional responses in intact neutrophils.

Activation of neutrophils with chemoattractants results in actin polymerization, followed by a rapidly oscillating actin polymerization/depolymerization response [41 ]. Based on our results showing peroxynitrite inhibition of actin polymerization/depolymerization in vitro, we hypothesized that peroxynitrite would inhibit this response in chemoattractant-stimulated neutrophils. As treatment of neutrophils with IL-8 has been shown previously to induce a transient polymerization of actin in neutrophils [41 ], we analyzed the effects of peroxynitrite on this response. Consistent with our in vitro studies, pretreatment of intact neutrophils with SIN-1 resulted in a concentration-dependent inhibition of the IL-8-induced actin polymerization (Fig. 9A ). In addition, the presence of 25 mM NaHCO3 enhanced the inhibitory effect, indicating a role for tyrosine nitration in the inhibitory effect.



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  		Figure 9. Inhibition of neutrophil actin polymerization and migration by SIN-1. Human neutrophils (107/ml) were incubated for 2 h in HBSS (open bars) or HBSS + 25 mM NaHCO3 (solid bars) containing the indicated concentrations of SIN-1. (A) Treated neutrophils were stimulated with 10-8 M IL-8, fixed, and stained with BODIPY-phallacidin, as described. Stained cells were analyzed by flow cytometry, and the results are expressed as % of control actin polymerization in buffer-treated cells. (B) Migration of treated neutrophils was measured in a transwell assay with control buffer or 10-7 M LTB4 in the lower wells, as described. The results are expressed as % of the maximum response seen in control LTB4-treated cells. In both panels, the results are expressed as mean ± SEM and represent three independent experiments. Statistically significant differences (#, P<0.01; *, P<0.001) between buffer control (0 mM) and SIN-1-treated samples are indicated.

 
Neutrophil migration requires actin polymerization/depolymerization [42 ]. Therefore, we analyzed the effect of SIN-1-generated peroxynitrite on cell migration. As shown in Figure 9B , migration toward LTB4 was inhibited in a concentration-dependent manner, with ~50% inhibition at 1.2 mM SIN-1 and nearly total inhibition at 5 mM. Again, SIN-1 treatment in the presence of NaHCO3 resulted in a substantial enhancement of the inhibitory effect such that migration was completely inhibited at the lowest concentration of SIN-1 (0.6 mM) and was lower than the negative control (no chemoattractant), suggesting that even chemokinesis was inhibited under these conditions.

The neutrophil cytoskeleton plays a key role in the process of phagocytosis [43 ]; therefore, we analyzed the effect of peroxynitrite on this important neutrophil function. As shown in Figure 10A , the ability of SIN-1-treated human neutrophils to phagocytose bacteria was significantly inhibited (~50%) at the lowest concentration of SIN-1 (0.6 mM), and control cells phagocytosed normally. It is interesting that no significant difference was observed for samples with added NaHCO3, suggesting inhibition was maximal with SIN-1 alone. The results were essentially the same for phagocytosis of S. aureus (Fig. 10A) or E. coli(not shown) BioParticles, indicating a general inhibitory effect that was not pathogen-specific. We also analyzed the ability of SIN-1-treated neutrophils to kill phagocytosed bacteria. At all time points, the level of bacterial killing in SIN-1-treated neutrophils was substantially (50–60%) lower than that observed in control cells (Fig. 10B) . Considering that neutrophils treated with 5 mM SIN-1 had ~40% of the phagocytic capability of control cells (Fig. 10A) , it appears that the relative level of killing of phagocytosed bacteria is similar in control and SIN-1-treated cells. Thus, SIN-1-treated neutrophils appear to be able to kill bacteria normally; however, they killed fewer total bacteria than control cells, which is consistent with the decreased level of phagocytosis in SIN-1-treated cells (Fig. 10B) .



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  		Figure 10. Effect of SIN-1 on neutrophil phagocytosis and bacterial killing. (A) Human neutrophils (107/ml) were incubated for 2 h in HBSS (open bars) or HBSS + 25 mM NaHCO3 (solid bars) containing the indicated concentrations of SIN-1. The treated cells were incubated with fluorescein-labeled S. aureus BioParticles, and intracellular fluorescence was measured as described. The level of phagocytosis is expressed as relative mean fluorescence above control (reagent blank). The results are expressed as mean ± SEM and represent three independent experiments. Statistically significant differences (*, P<0.001) between control (0 mM) and SIN-1-treated samples are indicated. (B) Human neutrophils (107/ml) were incubated at 25°C for 2 h in HBSS ({blacksquare}) or HBSS containing 5 mM SIN-1 ({circ}). The treated cells were mixed with opsonized S. aureus (1:1), and bacterial killing was determined as described. The results are expressed as the number of bacteria killed adjusted for bacterial growth at each time point. The results are representative of three independent experiments with three to five different dilutions plated and counted for each time point.

 
The human neutrophil NADPH oxidase is intimately associated with the membrane cytoskeleton [40 ] and has been shown to be modulated by actin [44 ]. Consistent with an association of actin and the NADPH oxidase, we found that the fMLF-activated respiratory burst of neutrophils was inhibited by SIN-1 treatment in a concentration-dependent manner, with an IC50 of ~0.6 mM SIN-1 and significant inhibition of oxidase activity at 5 mM SIN-1 (Fig. 11A ). Furthermore, inclusion of 25 mM NaHCO3 dramatically enhanced the inhibitory effect, again suggesting that nitrotyrosine formation played an important role in inhibiting this response.



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  		Figure 11. Effect of SIN-1 on neutrophil respiratory burst activity. (A) Human neutrophils (107/ml) were treated for 2 h with the indicated concentrations of SIN-1 in HBSS (open bars) or HBSS + 25 mM NaHCO3 (solid bars) and were stimulated with fMLF. O2– generation was determined using a cytochrome c-based assay, as described. The results are expressed as the % of maximal SOD-inhibitable O2– generation observed in control fMLF-treated cells (mean±SEM) and are representative of three independent experiments. Statistically significant differences (**, P<0.05; #, P<0.01; *, P<0.001) between control (0) and SIN-1-treated samples are indicated. (B) Cytochrome c (200 mM stock) was incubated with the indicated concentrations of SIN-1 for 30 min (open bars) and 1 h (cross-hatched bars) at 25°C. Aliquots were added to duplicate microtiter plate wells (0.1 mM final cytochrome c) containing 1.5 mM X and 20 mU/ml XO in 50 mM Hepes/125 mM NaCl (pH 7.4), and O2– production was monitored at 550 nm, as described. To verify O2– production, representative wells containing 200 U SOD are shown for cytochrome c samples treated with 0.5 mM SIN-1. The results are expressed as % of control activity detected by untreated cytochrome c (mean±SEM) and are representative of three independent experiments.

 
To verify that SIN-1 was not directly altering the ability of cytochrome c to detect O2–, we preincubated cytochrome c with a range of SIN-1 concentrations and analyzed the ability of SIN-1-treated cytochrome c to detect O2– generated by X/XO. As shown in Figure 11B , SIN-1 treatment had no significant effect on the ability of cytochrome c to detect O2–. Actual production of O2– by the X/XO system was confirmed by showing that SOD could completely inhibit cytochrome c reduction (Fig. 11B) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Peroxynitrite is a potent, biological oxidant that has been shown to play a role in free radical-mediated tissue injury [4 ]. Furthermore, the ability of peroxynitrite to oxidize or nitrate tyrosine residues on a variety of proteins appears to contribute to its cytotoxic effects (reviewed in refs. [45 , 46 ]). Previously, peroxynitrite has been shown to oxidize or nitrate and thereby inactivate a number of proteins, including Mn-SOD [14 ], aconitase [47 ], glutamine synthetase [48 ], alcohol dehydrogenase [49 ], creatine kinase [50 ], and metalloproteinase-1 inhibitor [51 ]. In the present studies, we show that peroxynitrite modifies actin, primarily by nitration, resulting in an inhibition of G-actin polymerization and depolymerization of F-actin. In a cell, the net result would be an increased level of actin monomers, a reduced level of actin filaments, and an impaired ability to modulate the actin cytoskeleton.

Actin dynamics play an essential role in a number of distinct neutrophil functions, including chemotaxis, phagocytosis, secretion, and activation of the respiratory burst [52 ]. For example, a number of studies have shown that chemoattractants stimulate actin polymerization in neutrophils (reviewed in ref. [53 ]). Actin reorganization has also been shown to play an important role in neutrophil surface morphology [54 ], priming [18 ], and adhesion [55 ]. Previously, we found that the neutrophil NADPH oxidase localized to actin-rich membrane domains [40 ] and that many of the respiratory burst components became associated with the cytoskeleton [56 ]. More recently, Al-Mohanna and Hallett [57 ] reported that actin polymerization associated with chemoattractant stimulation played a role in neutrophil NADPH oxidase activation, and Granfeldt and Dahlgren [58 ] demonstrated that an intact actin cytoskeleton was required for prolonged respiratory burst activity during neutrophil phagocytosis. These findings have been confirmed in a number of studies, including recent reports by Tamura and coworkers [44 , 59 ] showing that actin depolymerizing agents inhibited the neutrophil NADPH oxidase in a cell-free assay and that one of the NADPH oxidase proteins directly interacted with actin in vitro. Based on the importance of actin dynamics in neutrophil functional responses and the ability of SIN-1-generated peroxynitrite to inhibit actin polymerization/depolymerization in vitro, we further investigated whether peroxynitrite would alter a number of actin-dependent neutrophil functions. As shown here, peroxynitrite inhibited neutrophil actin polymerization, migration, phagocytosis, and NADPH oxidase activity ex vivo. Based on these data and previous studies showing that peroxynitrite could inhibit a fifth actin-dependent neutrophil function, ß-glucuronidase release [60 ], as well as NADPH oxidase activity [60 , 61 ], we now provide a global, mechanistic framework to explain these observations (i.e., peroxynitrite-mediated actin nitration inhibits actin function, which interferes with neutrophil actin-dependent responses). Thus, it is clear that exposure of neutrophils to peroxynitrite in the inflammatory milieu would have a significant impact on the responsiveness of these cells, in part, through inhibition of actin-dependent functions. Note that Sato et al. [62 ] recently reported that peroxynitrite could directly alter the chemotactic activity of IL-8 via direct nitration of IL-8. Thus, peroxynitrite can have profound effects on the inflammatory agent as well as on the inflammatory cell, as shown here.

Previous studies have suggested that peroxynitrite can induce apoptosis in neutrophils [63 , 64 ]; however, this effect is highly dependent on the concentration of peroxynitrite and incubation time. For example, Blaylock et al. [63 ] reported significant increases in neutrophil annexin V staining only after >=8 h incubation with 1 mM SIN-1. Similarly, Ward et al. [64 ] found that significant increases in neutrophil apoptosis occurred only after >=6 h incubation with 1 mM SIN-1 or after >=4 h with >100 µM peroxynitrite. In the present studies, neutrophils were treated for <=2 h with SIN-1; therefore, very little apoptosis would be expected. In addition, significant effects on neutrophil function were observed at much lower SIN-1 or peroxynitrite concentrations than those required to induce apoptosis. Thus, we conclude that the effects of peroxynitrite on actin-dependent neutrophil functions reported here are not a result of apoptosis. Indeed, we have analyzed neutrophil apoptosis under similar incubation conditions and confirmed that no significant increases in apoptosis above spontaneous apoptosis occurred until >=6 h and with >=100 µM peroxynitrite (data not shown). At lower peroxynitrite concentrations (50 µM), induction of apoptosis required even longer incubation periods (>=8 h).

Currently, there is strong evidence indicating that peroxynitrite is formed from NO and superoxide [65 ] and that peroxynitrite nitrates tyrosine residues on various cellular proteins in vitro and in vivo [46 ]. Although peroxynitrite has been shown to cause other protein modifications, such as oxidation of amino acids to form carbonyl derivatives and dityrosine cross-links [66 ], we observed little or no dityrosine or carbonyl formation in SIN-1-treated actin at any of the SIN-1 concentrations tested (0–5 mM). In addition, blocking tyrosine nitration by PNU-101033E completely abrogated the inhibitory effects of SIN-1 on actin polymerization/depolymerization. Thus, we conclude that the effects of peroxynitrite on actin function were primarily a result of nitration. This conclusion is consistent with studies of Tien et al. [35 ], who reported that peroxynitrite does not contribute significantly to carbonyl formation or methionine oxidation at physiological pH and CO2 concentrations.

The molecular basis for enzyme inactivation by tyrosine nitration is still not well understood; however, Yamakura et al. [67 ] found that inactivation of human Mn-SOD by peroxynitrite was caused by exclusive nitrotyrosine formation at Tyr34. Thus, nitration of a single key residue in or near the active site of an enzyme is sufficient to disrupt biological activity. Accordingly, it seems reasonable that only a low level of tyrosine nitration, as was observed in SIN-1-treated cells in our system and as is present in inflammatory tissues in vivo [4 ], would be required to alter a protein’s function. It is interesting that analysis of the structure of actin indicates that 16 tyrosine residues are present in human and rabbit actin and that some tyrosines are located in key functional regions. For example, Tyr53 plays a role in stabilizing the DNase I-binding loop (His40–His50) within actin subdomain 2, and fluorescein labeling of Tyr53 has been shown to block actin polymerization. Tyr69 is also located in actin subdomain 2. This subdomain is involved in intermonomer interactions within the actin filament [68 ] and is the region that undergoes a conformational change during ATP hydrolysis [69 ]. Tyr143 is located in the hydrophobic pocket of actin subdomain 1, a region involved in profilin binding, and is displaced by tetramethylrhodamine-5-maleimide, which covalently modifies actin at Cys374 and blocks actin polymerization [69 ]. Tyr306 is located in actin subdomain 3, forms part of the nucleotide-binding pocket, and has been shown to be close to the adenine base [70 ]. Thus, it is possible that nitration of only one or two key tyrosine residues in actin might significantly alter actin function to the degree reported here. Previously, Crow et al. [71 ] suggested that the presence of neighboring acidic residues plays a role in increasing the susceptibility of these tyrosines to nitration. In the primary sequence, a number of actin tyrosine residues are flanked by glutamate and/or aspartate residues (Tyr166, -188, -240, -362); however, the G-actin crystal structure indicates that protein folding also brings Try69 into close proximity with Glu72 [69 ]. The close proximity of the Glu72 carboxyl group to Tyr69 would then help stabilize charged intermediates in the nitration reaction [71 ].

Peroxynitrite is generated at sites of inflammation [4 ]; therefore, it is likely that neutrophils do encounter significant levels of peroxynitrite that could modify actin and other neutrophil proteins. In the present studies, we show a concentration-dependent inhibition of actin polymerization, and significant inhibition begins at 10 µM and an IC50 of ~45 µM peroxynitrite. As a result of the short half-life of peroxynitrite (~1 s at physiological pH), bolus peroxynitrite would be almost completely degraded within seconds, and Beckman et al. [72 ] proposed that the actual effective concentration of peroxynitrite should be expressed as a "concentration x time product." For a bolus peroxynitrite concentration of 45 µM, therefore, the effective steady-state concentration of peroxynitrite would be ~58 nM for the 20-min incubation period, which is well within physiological levels. However, addition of bolus peroxynitrite required pH adjustments, which may have some undetermined effects on actin function. Therefore, we used a peroxynitrite donor, SIN-1, which has been shown to generate lower levels of peroxynitrite over an extended period of time and may represent a more physiologically relevant application of this oxidant [73 ]. Consistent with our data using bolus peroxynitrite, SIN-1 treatment also effectively inhibited actin polymerization in a concentration-dependent manner (IC50 of ~0.9 mM SIN-1 with low NaHCO3 and 0.1 mM SIN-1 with high NaHCO3). Based on an estimated yield of 10 µM min peroxynitrite from 1 mM SIN-1 at 37°C and pH 7.4 [74 ], the IC50 in SIN-1-treated samples would be ~69 and 8 nM peroxynitrite for the 20-min incubation period in samples containing low and high NaHCO3, respectively. Thus, the concentration range of peroxynitrite required to inhibit actin polymerization was similar in samples treated with bolus peroxynitrite or SIN-1. Given that activated human neutrophils can generate up to 0.2 nmol/min peroxynitrite, with estimated local concentrations of ~100 µM [8 ], it is likely that the steady-state levels of peroxynitrite used here would be encountered by neutrophils at sites of inflammation where significant concentrations of NO and O2– are produced.

Peroxynitrite has been shown to be bactericidal [75 ]; however, the mechanisms are not well understood. Studies by Hausladen and Fridovich [47 ] found that aconitase, one of the citric acid cycle enzymes, was a target for peroxynitrite in E. coli, suggesting that enzyme inactivation played a role in peroxynitrite toxicity. As microbial pathogens use actin-based motility mechanisms [76 ], they would also be susceptible to the effects of peroxynitrite on actin function. Consequently, peroxynitrite produced at sites of inflammation could impair motility and possibly inhibit the ability of microbes to migrate into neighboring tissues or evade phagocytic cells. This may represent a novel microbicidal mechanism involving peroxynitrite; however, further studies will be necessary to evaluate this possibility. In any case, it is clear that the ability of peroxynitrite to inhibit actin dynamics would have a significant effect on any actin-dependent cellular process.


   ACKNOWLEDGEMENTS
 
This work was supported in part by NIH RO1 HL66575 (M. T. Q.), NIH RO3 AG19386 (T. T. R.), and the Montana State University Agricultural Experimental Station. This is manuscript 2002-33 from the Montana Agricultural Experiment Station, Montana State University-Bozeman. We thank Pharmacia & Upjohn Co. for generously providing PNU-101033E for these studies.

Received August 20, 2002; revised November 22, 2002; accepted November 28, 2002.


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 RESULTS
 DISCUSSION
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